Fig. 5: Double knockout of outward-oriented CBS3 and CBS5 elements downregulates HOXD13 gene expression.

a CTCF and H3K27ac ChIP-seq profiles of the HOXD centromeric regulatory TAD (C-DOM) in wild-type (WT) and in CBS3 and CBS5 double knockout (ΔCBS3 + 5) homozygous single-cell clones. The left boundary (red-dotted rectangle) comprises a cluster of CTCF sites, CBS1-6, of which CBS3 and CBS5 are in the outward orientation. Arrowheads indicate CBS elements with orientations. b Close-up of CTCF ChIP-seq profiles at the C-DOM left boundary marked by red dotted rectangle in (a). Note the abolishment of CTCF peaks at the locations of CBS3 and CBS5 in the three single-cell deletion clones. c Close-up of CTCF and H3K27ac ChIP-seq profiles at the HOXD cluster in (a). Note that the five reverse-oriented CBS elements are associated with HOXD13 but not with other HOXD genes. d 4C profiles using HOXD13 as an anchor in ΔCBS3 + 5 single-cell clones compared to WT clones, showing increased chromatin interactions with the left boundary and decreased chromatin interactions with the H3K27ac-enriched GT2 or LNPK element upon double knockout. e, f 4C profiles using CBS2 (e) or CBS4 (f) as an anchor confirming increased chromatin interactions with HOXD13. g Expression levels of HOXD genes in three ΔCBS3 + 5 single-cell clones compared to WT clones. Data as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001; two-tailed Student’s t test. For each deletion clone, n = 2 biologically independent samples; for their WT controls, n = 4. Source data are provided as a Source Data file. h A boundary model illustrating the role of outward-oriented CTCF sites (CBS elements) in intra-TAD chromatin contacts and gene regulation. TAD boundary normally contains clustered CTCF sites with complex orientations of both divergence and convergence. The outward-oriented CTCF sites function as an important barrier for outside cohesin to ensure proper and balanced intra-TAD chromatin interactions and gene expression.